4092
The Journal of Experimental Biology 213, 4092-4098
© 2010. Published by The Company of Biologists Ltd
doi:10.1242/jeb.045088
Characterization of mechanisms for Ca2+ and HCO3–/CO32– acquisition for shell
formation in embryos of the freshwater common pond snail Lymnaea stagnalis
Sue C. Ebanks1, Michael J. O’Donnell2 and Martin Grosell1
1
University of Miami, Rosenstiel School of Marine and Atmospheric Science, Division of Marine Biology and Fisheries,
4600 Rickenbacker Causeway, Miami, FL 33149, USA, 2Department of Biology, McMaster University, 1280 Main Street West,
Hamilton, Ontario L8S 4K1, Canada
Accepted 6 September 2010
SUMMARY
The freshwater common pond snail Lymnaea stagnalis produces embryos that complete direct development, hatching as shellbearing individuals within 10days despite relatively low ambient calcium and carbonate availability. This development is impaired
by removal of ambient total calcium but not by removal of bicarbonate and/or carbonate. In this study we utilized pharmacological
agents to target possible acquisition pathways for both Ca2+ and accumulation of carbonate in post-metamorphic, shell-laying
embryos. Using whole egg mass flux measurements and ion-specific microelectrode analytical techniques, we have demonstrated
that carbonic anhydrase-catalyzed hydration of CO2 is central in the acquisition of both shell-forming ions because it provides the
hydrogen ions for an electrogenic vacuolar-type H+-ATPase that fuels the uptake of Ca2+ via voltage-dependent Ca2+ channels and
possibly an electrogenic Ca2+/1H+ exchanger. Additionally, CO2 hydration provides an endogenous source of HCO3–. Thus,
hydration of endogenous CO2 forms HCO3– for calcification while hydrogen ions are excreted, contributing to continued Ca2+
uptake, as well as creating favorable alkaline internal conditions for calcification. The connections between Ca2+ and HCO3–
acquisition mechanisms that we describe here provide new insight into this efficient, embryonic calcification in freshwater.
Key words: pulmonate snail, metamorphosis, ion-selective microelectrode, ion transport, calcification, embryonic development.
INTRODUCTION
Embryos of the freshwater common pond snail Lymnaea stagnalis
complete direct development, emerging as shell-bearing hatchlings
approximately 10days post-oviposition in a low-ionic strength,
minimally buffered environment. In the absence of ambient total
calcium (Ca), they tend to develop at a retarded rate relative to
controls but grow and hatch at least as well as controls when reared
in carbonate-free water (Ebanks et al., 2010). Although they are
provided with maternal stores of calcium and carbonate in the
perivitelline fluid and within the gelatinous matrix (tunica interna)
of the egg mass, they begin to rely on Ca2+ from the surrounding
medium as maternal stores in the perivitelline fluid and tunica
capsulis become depleted, at approximately day5 post-oviposition
(Ebanks et al., 2010). At this point in embryonic development,
embryos increase oxygen consumption and total CO2 retention
(Baldwin, 1935). Metamorphosis and shell formation have been
observed for Lymnaea palustris (Morrill, 1982), L. stagnalis (Ebanks
et al., 2010) and another freshwater pulmonate snail Biomphalaria
glabrata (Bielefeld and Becker, 1991; Marxen et al., 2003). The
post-metamorphic Ca2+ requirements seem to be met entirely by
active uptake, but the carbonate requirements can apparently be
accommodated through both uptake and endogenous sources or by
endogenous sources exclusively (Ebanks et al., 2010).
The idea of Ca2+ uptake in freshwater being by active uptake
through Ca2+ channels has been presented, studied and reviewed
from many angles and by many researchers but mostly in freshwater
trout (Flik et al., 1995; Marshall et al., 1992; Perry and Flik, 1988;
Shahsavarani and Perry, 2006). An electrochemical gradient for
Ca2+, established by basolateral Ca2+-ATPase and Na+/K+-ATPase
activity, and further facilitated and maintained by an apical,
outwardly directed H+ pump provides the necessary gradient to allow
for Ca2+ uptake under these conditions (Perry et al., 2003). Hydrogen
ions supplied to this pump are mostly from hydration of CO2. In
addition to acid excretion by the H+ pump, protons can be extruded
in exchange for monovalent, as well as divalent, cations via Na+/H+
exchangers (NHE) by electroneutral or electrogenic exchange (for
a review, see Ahearn et al., 2001). Recent work by Okech et al.
(Okech et al., 2008) indicated cation exchange for hydrogen ions
can be coupled with vacuolar H+-ATPase (V-ATPase) activity on
electrogenic Na+/H+ antiporters. A further point of consideration
that is important to this study is that 2Na+/1H+ electrogenic
exchange, which is apparently unique to invertebrates, can
accommodate divalent cations, including Ca2+, in place of the two
Na+ (Ahearn et al., 1994).
Our previous observations of unimpeded growth under HCO3–free conditions led to subsequent experiments examining the
transport of acid and base equivalents under calcifying, postmetamorphic conditions. We found that the onset of apparent net
titratable alkalinity uptake/acid extrusion was concurrent with
metamorphosis, the onset of Ca2+ uptake and shell formation. Also,
Ca2+ uptake was significantly greater and apparent net titratable
alkalinity uptake/acid extrusion was significantly reduced, but not
inhibited, under HCO3–-free conditions (Ebanks et al., 2010). These
findings motivated the series of experiments to determine the
possible sources of carbonate for shell formation that are described
in this paper.
Considering that carbonic anhydrase-mediated CO2 hydration
produces HCO3–, this was a logical starting point. The possibility
of endogenous bicarbonate contributing to calcification in the
embryos (Baldwin, 1935) and adults (Greenaway, 1971) of this
species has been suggested previously in the literature. However,
the point of development at which this pathway is activated has not
THE JOURNAL OF EXPERIMENTAL BIOLOGY
Embryonic snail shell ion acquisition
4093
been studied until recently (Ebanks et al., 2010). Also, the possible
role of the proton, resulting from CO2 hydration and CaCO3
formation, in the acquisition of the essential shell-forming ions by
these embryos remains to be evaluated. Therefore, the objectives
of this study were to use a pharmacological approach in which flux
was measured on the scale of the whole egg mass and the microscale
of single eggs to determine the acquisition pathways for shellforming ions in post-metamorphic embryos of the freshwater
common pond snail Lymnaea stagnalis. Additionally, we targeted
the possible relationship between protons produced by carbonic
anhydrase activity and Ca2+ uptake. Each of the proposed pathways
was evaluated on whole egg mass and at microscale levels when
possible.
electrogenic H+ excretion via the H+ pump. Bafilomycin (SigmaAldrich), a proton-pump inhibitor, was dissolved in DMSO for a
final concentration of 1moll–1 bafilomycin and 0.1% DMSO in
each flux chamber. Day-7 egg masses were scored for viability and
placed in individual flux chambers for a 24-h incubation, after which
we determined net Ca2+ and titratable alkalinity flux as described
below. Also, because of the potential for direct exchange of Ca2+
for H+ on an apical invertebrate-specific cation/1H+ exchanger, we
completed a 24-h flux experiment in which day-7 egg masses were
treated with 100moll–1 of the NHE-specific amiloride analogue,
ethylisopropylamiloride (EIPA; Sigma-Aldrich) dissolved in a final
concentration of 0.1% DMSO.
MATERIALS AND METHODS
Test animals
Egg masses of the common pond snail Lymnaea stagnalis (L.) were
collected from our laboratory snail culture at 24-h intervals, with
the day of collection considered day0 post-oviposition, for age
determination. Embryos were incubated at room temperature
(20–22°C) in aerated, dechlorinated Miami-Dade County tap water
(Grosell et al., 2007). Whole, intact egg masses were selected and
scored for viability in accordance with criteria outlined in Ebanks
et al. (Ebanks et al., 2010) prior to use in experimental procedures.
With an exception of the niphedipine, verapamil and bafilomycin
experiments using the ion-specific microelectrodes, all experiments
were completed on intact egg masses as defined in Ebanks et al.
(Ebanks et al., 2010).
The procedures for determination of flux rates were in accordance
with those outlined by Ebanks et al. (Ebanks et al., 2010). Briefly,
samples of pre- and post-flux media were collected for each
treatment and Ca2+ net flux was determined by using the difference
in [Ca]total in initial and final flux media following the 24-h flux.
Samples were measured by flame atomic absorption
spectrophotometry (Varian SpectrAA220, Mulgrave, Victoria,
Australia) and flux rates were calculated from the change in
[Ca]total, mass of egg mass (g), and elapsed time (h). Net titratable
alkalinity flux was calculated from the change in titratable alkalinity
as determined by double endpoint titration (Ebanks et al., 2010),
mass of egg mass (g) and elapsed time (h). Final values for both
Ca2+ and titratable alkalinity net flux parameters are presented as
molg–1h–1.
Whole egg mass flux experiments
Ca2+-selective and H+-selective microelectrodes
Analytical procedures for whole egg mass experiments
2+
Three Ca channel blockers were used independently to determine
the type and the role of Ca2+ channels that may be involved.
Nifedipine, which is a voltage-dependent L-type Ca2+ channel
blocker, was applied as was verapamil, another voltage-dependent
L-type Ca2+ channel blocker and 1-adrenoceptor antagonist.
Lanthanum, an indiscriminate Ca2+ channel blocker was also tested.
Day7 egg masses were scored for normal development and placed
in Miami-Dade tap water that was either drug-free control (0.1%
dimethylsulphoxide; DMSO) or treated for 24h with either
10moll–1 nifedipine (Sigma-Aldrich, St Louis, MO, USA),
verapamil
hydrochloride
(Sigma-Aldrich),
10moll–1
10moll–1La3+ or 100moll–1La3+ as LaCl3. Initial and final water
samples were collected for Ca2+ and titratable alkalinity (TA) net
flux determinations, which were completed following the protocol
for double-endpoint titration described by Ebanks et al. (Ebanks et
al., 2010), and are reported in molg–1h–1. Considering that
carbonate necessary for calcification could be from aquatic sources
or endogenous processes (Ebanks et al., 2010), a multi-pronged
experimental approach was required. The role of carbonic anhydrase
(CA) in the production of endogenous bicarbonate via CO2 hydration
was evaluated using a lipophilic carbonic anhydrase inhibitor,
ethoxzolamide (ETOX; Sigma-Aldrich), dissolved in DMSO.
Controls (0.1% DMSO) and 100moll–1 ETOX-exposed egg
masses were incubated for 24h; water samples were taken before
and after incubation for Ca2+ and TA net flux determinations. In
addition to HCO3–, CA-catalyzed hydration of CO2 also produces
equal quantities of H+, which are counterproductive to shell
formation and must be excreted to make CO2 hydration a viable
source of CO32– for shell formation. Thus we evaluated the role of
V-ATPase (a H+ pump) in the removal of protons and possibly Ca2+
uptake, considering that apical Ca2+ channels may be driven by an
inward-directed electrochemical gradient fueled in part by
Microelectrodes used to measure Ca2+ and H+ gradients in the
unstirred layer next to the surface of egg masses and isolated eggs
were pulled from unfilamented 1.5mm borosilicate capillary glass
(A-M Systems, Carlsborg, WA, USA), silanized by exposure to
dimethyldichlorosilane vapor at 200°C for 60min and stored over
silica gel until use. Ca2+-selective microelectrodes were backfilled
with 100mmoll–1 CaCl2 and tip-filled with a 300–500m column
of Ca2+ ionophore I, cocktail A (Fluka, Buchs, Switzerland). H+selective microelectrodes were backfilled with a solution of
100mmoll–1 NaCl and 100mmoll–1 sodium citrate at pH6, then
tip-filled with a 300–500m column of H+ ionophore I, cocktail B
(Fluka). H+ microelectrodes calibrated in solutions buffered with
10mmoll–1 Hepes gave Nerstian slopes of 58–60mV per pH unit.
Ca2+ and H+ fluxes: microscale measurements using the
scanning ion electrode technique
Transport of calcium ions or protons into or out of egg masses or
isolated eggs produces gradients in Ca2+ or H+ concentration in the
unstirred layer adjacent to the surface of the preparation. These
gradients can be calculated from the voltages recorded by an ionselective microelectrode moved between two points within the
unstirred layer. Calcium flux can then be calculated from the Ca2+
concentration gradients using Fick’s law. Measurement of fluxes in
this way is the basis of the scanning ion electrode technique (SIET),
which allows fluxes to be repeatedly measured in near real-time at
sites along the length of an egg mass or an isolated egg (Ebanks et
al., 2010).
SIET measurements were made using hardware from
Applicable Electronics (Forestdale, MA, USA) and automated
scanning electrode technique (ASET) software (version 2.0)
from Science Wares Inc. (East Falmouth, MA, USA). Egg masses
or isolated eggs were placed in a 35mm diameter Petri dish filled
THE JOURNAL OF EXPERIMENTAL BIOLOGY
4094 S. C. Ebanks, M. J. O’Donnell and M. Grosell
DC CB ⫻ 10(DV/S) – CB,
(1)
where DC is the Ca concentration difference between the two limits
of the excursion distance (in molcm–3), CB is the background Ca2+
concentration in the bathing medium, DV is the voltage gradient (mV)
and S is the slope of the electrode between 0.1 and 1mmoll–1 Ca2+,
bracketing the Ca2+ concentration in Miami-Dade County tap water.
Although ion-selective microelectrodes measure ion activity and not
concentration, data can be expressed in terms of concentrations if it
is assumed that the ion activity coefficient is the same in calibration
and experimental solutions. Expression of data as concentrations aids
comparison of Ca2+ microelectrode data with those of techniques
such as atomic absorption spectrophotometry, which measures Ca2+
concentration. Concentrations can be converted to activities by
multiplying by the activity coefficient (0.81 for Miami-Dade tap
water). The Ca2+-selective microelectrodes used in conjunction with
SIET can resolve changes in voltage exceeding the magnitude
measured at the reference sites of ~10V, corresponding to changes
in Ca2+ activity of 0.08% in Miami-Dade tap water. Typical signals
of 300V correspond to changes in Ca2+ activity of 2.4%.
Concentration differences were used to determine the Ca2+ flux
using Fick’s law of diffusion:
2+
JCa DCa(DC / DX),
(2)
where JCa is the net flux in molcm–2s–1, DCa is the diffusion
coefficient of Ca2+ (1.19⫻10–5cm2s–1), DC is the Ca2+ concentration
gradient and DX is the excursion distance between the two points
(cm).
Calculation of H+ fluxes requires knowledge of the buffers that
are present (e.g. Donini and O’Donnell, 2005). Ideally, a non-volatile
buffer such as Hepes is used. However, because we wished to use
the same Miami-Dade tap water as used for the whole egg mass
flux experiments, we did not modify the composition of the water
and therefore we present H+ gradients as microelectrode voltage
rather than proton flux in the Results.
Statistical analyses
Comparisons were completed using either t-test or an ANOVA with
contingent significant results evaluated as pairwise comparisons
following the Holm–Sidak method after an analysis for whether the
datasets were normally distributed. Statements of significance were
reserved for those comparisons for which P<0.05.
RESULTS
Absolute flux rates of egg masses of similar age varied from batch
to batch even under similar conditions. The reason for this variation
is unknown but underscores the importance of performing control
and treatment experiments simultaneously and on egg masses from
the same batch as was done in the present and previous study
(Ebanks et al., 2010). Results from our multi-pronged
pharmacological approach indicated that voltage-dependent Ca2+
channels, H+-pump activity and Na+/H+ antiport function in
conjunction with CA activity allow the post-metamorphic embryonic
snail to complete calcification and shell formation in freshwater.
The two voltage-dependent Ca2+ channel blockers had different
effectiveness against net Ca2+ uptake with nifedipine being the more
effective blocker when examined by net Ca2+ flux measurements
and SIET techniques (Figs1 and 2). Verapamil did not significantly
block net Ca2+ transport at 10moll–1 but 10moll–1 nifedipine did
in whole egg mass experiments (Fig.1). Scanning Ca2+-selective
microelectrode analysis showed that both voltage-dependent Ca2+channel blockers reduced Ca2+ transport at 10moll–1 but that
100moll–1 verapamil completely blocked net Ca2+ uptake (Fig.2).
Neither drug influenced net titratable alkalinity flux (data not
shown). Lanthanum blocked net Ca2+ uptake at 100moll–1 but not
at 10moll–1 in whole egg mass experiments (Fig.3). There was
also a significant increase in apparent net titratable alkalinity
uptake/acid extrusion in the presence of 100molLa3+(data not
shown). Microelectrode experiments revealed a tendency for reduced
Ca2+ uptake with 100moll–1La3+ but just below statistical
significance (Fig.4).
Using 100moll–1 ETOX to evaluate the role of CA-catalyzed
hydration of CO2 on whole egg mass flux rates, we observed a
significant reduction in both net Ca2+ uptake and apparent titratable
alkalinity uptake/acid extrusion (Fig.5). This link between CO2
hydration and Ca2+ uptake led to the testing of the possible role of
the apical H+ pump and Na+/H+ exchange by evaluating the
inhibitory effects of bafilomycin and EIPA at concentrations of
1moll–1 and 100moll–1, respectively, on Ca2+ and titratable
0.4
Ca2+ net flux (µmol g–1 h–1)
with 5ml of artificial Miami-Dade tap water. Reference electrodes
were made from 10cm lengths of 1.5mm borosilicate glass
capillaries that were bent at a 45deg angle, 1–2cm from the end,
to facilitate placement in the sample dish. Capillaries were filled
with boiling 3moll–1 KCl in 3% agar and connected to the ground
input of the Applicable Electronics amplifier headstage through
a Ag/AgCl half cell.
Ca2+-selective microelectrodes for use with SIET were calibrated
in 0.1, 1 and 10mmoll–1 Ca. The electrodes showed Nerstian slopes
of 28–30mV per 10-fold change in Ca2+ concentration. All scans
were performed in artificial Miami-Dade water with a [Ca]total of
400moll–1. The Ca2+-selective microelectrode was initially placed
5–10m from the surface of the egg mass or isolated egg. The
microelectrode was then moved a further 50m away, perpendicular
to the egg mass surface. The ‘wait’ and ‘sample’ periods at each
limit of the 50m excursion distance were 4.5 and 0.5s, respectively.
Voltage differences across this excursion distance were measured
three times at each of the four to six sites along the surface of each
egg mass or isolated egg. There were no significant differences in
measurements taken at different sites around the egg masses or eggs,
thus measurements were pooled for each sampling period. Voltage
differences were corrected for electrode drift measured at a reference
site 10–20mm away from the egg mass. Voltage differences at
measurement sites and at reference sites were typically >300V
and <10V, respectively. Voltage differences (DV) were converted
to the corresponding Ca2+ concentration difference by the following
equation (Donini and O’Donnell, 2005):
0.3
0.2
(10)
0.1
(6)
0
(8)
–0.1
–0.2
*
–0.3
–0.4
Control
Nifedipine
Verapamil
Fig.1. Net Ca2+ flux rate (molg–1h–1) under control conditions (0.1%
DMSO) and during pharmacological manipulations (10moll–1 verapamil or
10moll–1 nifedipine). Values are means ± s.e.m. Numbers in parentheses
are the numbers of egg masses examined. Ambient [Ca2+]648±7moll–1.
*, significantly different from control (P<0.05).
THE JOURNAL OF EXPERIMENTAL BIOLOGY
Embryonic snail shell ion acquisition
0.14
Ca2+ flux (µmol cm–2 h–1)
0.08
0.06
(6)
0.04
*
0.02
(10)
(6)
0
0.12
0.10
0.08
0.04
–0.02
0
Fig.2. Results of scanning Ca2+-selective microelectrode analysis of day-9
isolated eggs in control conditions (0.1% DMSO) and during treatment with
a voltage-dependent Ca2+ channel blocker (nifedipine or verapamil). Values
are means ± s.e.m. Numbers in parentheses are the numbers of eggs
scanned; five to seven measurements for each egg. *, significant reduction
from control values. Ambient [Ca2+]400moll–1.
alkalinity transport. Proton-pump inhibition resulted in inhibition
of net Ca2+ uptake (Fig.6A) and apparent net titratable alkalinity
uptake (equivalent to reduced acid secretion; Fig.6B). Scanning
Ca2+-selective microelectrode measurements revealed that
bafilomycin had a significant inhibitory effect of up to 80% on Ca2+
transport for post-metamorphic egg masses (data not shown).
Furthermore, and in agreement with whole egg mass fluxes, Ca2+
uptake was inhibited in isolated eggs containing later-stage day-11
to -13 embryos, treated with 1moll–1 bafilomycin (Fig.7A) and
H+ gradients were effectively reversed (Fig.7B) as measured with
ion-selective microelectrodes in the unstirred boundary layer.
Similar to the bafilomycin treatment, both Ca2+ (Fig.8A) and
titratable alkalinity (Fig.8B) net flux were reversed in whole egg
mass fluxes where flux medium was treated with 100moll–1EIPA.
DISCUSSION
The system for Ca2+ and carbonate acquisition in the freshwater
common pond snail Lymnaea stagnalis exhibited sensitivity to
several pharmacological agents, which indicate that both Ca2+ uptake
from the environment as well as endogenous HCO3– and/or CO32–
production work together directly and indirectly for the postmetamorphic embryo to acquire the necessary ions for calcification
in freshwater (Fig.9). Both the dihydropyridine nifedipine and the
phenylalkylamine verapamil are known to block L-type Ca2+
channels, with nifedipine displaying relatively greater specificity
and efficacy to L-type voltage-dependent Ca2+ channels (Hagiwara
and Byerly, 1981). These compounds inhibit Ca2+ channels in some
epithelia such as the distal convoluted tubule of the rabbit kidney
(Poncet et al., 1992) but not in others, such as the cortical thick
ascending limb (Nitschke et al., 1991). The model that we are
3
–0.1
(14)
(12)
*
Control
10 µmol l–1 100 µmol l–1
La3+
La3+
Fig.3. Net Ca2+ flux rates (molg–1h–1) in control conditions and during
treatment with 10moll–1 or 100moll–1 La3+ as LaCl3. Values are means
± s.e.m. Numbers in parentheses are the numbers of egg masses
examined. Ambient [Ca2+]762±0.3moll–1. *, significantly different from
control (P<0.05).
TA net flux (µmol g–1 h–1)
0
A
2
(7)
1
0
(8)
–1
–2
3
(12)
100 µmol l–1 La3+
Fig.4. Results of scanning Ca2+-selective microelectrode analysis of intact
day-9 to -10 egg masses in control conditions (0.1% DMSO) and during
treatment with a voltage-independent Ca2+ channel blocker (100moll–1
La3+ as LaCl3). Values are means ± s.e.m. Numbers in parentheses are
the numbers of eggs scanned; five to ten measurements for each egg.
Ambient [Ca2+]400moll–1.
0.1
Ca2+ net flux (µmol g–1 h–1)
Control
*
Control 10 µmol l–1 10 µmol l–1 100 µmol l–1
Nifedipine Verapamil Verapamil
–0.2
(6)
0.02
(5)
*
(6)
0.06
Ca2+ net flux (µmol g–1 h–1)
Ca2+ flux (µmol cm–2 h–1)
0.10
4095
*
B
2
1
0
(7)
(8)
*
–1
–2
Control
ETOX
Fig.5. Comparison of net Ca2+ (A) and titratable alkalinity (B) flux rates
(molg–1h–1) under control conditions (0.1% DMSO) and during
pharmacological manipulation with 100moll–1 ethoxzolamide (ETOX).
Values are means ± s.e.m. Numbers in parentheses are the numbers of
egg masses examined. Ambient [Ca2+]1388±79moll–1. *, significantly
different from control (P<0.05).
THE JOURNAL OF EXPERIMENTAL BIOLOGY
4096 S. C. Ebanks, M. J. O’Donnell and M. Grosell
0.3
A
1.2
1.0
0.8
0.6
0.4
0.2
(4)
0
(8)
Ca2+ flux (µmol cm–2 h–1)
Ca2+ net flux (µmol g–1 h–1)
1.4
*
–0.2
1.2
B
1.0
0.8
0.6
0.4
0.1
(5)
(5)
0
*
B
*
600
H+ gradient (µV)
TA net flux (µmol g–1 h–1)
1.4
0.2
–0.1
800
–0.4
A
(4)
0.2
(8)
0
–0.2
*
400
(6)
200
0
–200
–400
(6)
–600
–800
Control
Bafilomycin
–0.4
Control
Bafilomycin
Fig.6. Comparison of net Ca2+ (A) and titratable alkalinity (B) flux rates
(molg–1h–1) under control conditions (0.1% DMSO) and during treatment
with 1moll–1 bafilomycin. Values are means ± s.e.m. Numbers in
parentheses are the numbers of egg masses examined. Ambient
[Ca2+]407±1moll–1. *, significantly different from control (P<0.05).
Fig.7. Comparison of Ca2+ flux (A) and H+ gradients (B) within the unstirred
boundary layer of post-metamorphic isolated eggs at day7 and 8 in control
conditions (0.1% DMSO) and during treatment with 1moll–1 bafilomycin.
Eggs were scanned using a Ca2+- or H+-selective microelectrode under
control conditions, exposed to drug for 30–60min, then scanned in drugtreated medium (paired design). Values are means ± s.e.m. Numbers in
parentheses are the numbers of eggs examined. Ambient
[Ca2+]400moll–1. *, significantly different from control (P<0.05).
proposing here for post-metamorphic embryos compliments findings
of Greenaway (Greenaway, 1971) using radioisotopic techniques
on adults of the species. In the Greenaway model for adult Lymnaea,
blood, fresh tissues and shell are identified as the major
compartments between which Ca2+ is transported (Greenaway,
1971). In times of shell accretion, Ca2+ is taken up from the
surrounding medium or from food and into the blood for transport
to fresh tissues, particularly the mantle, for deposition as CaCO3
after combining with endogenously produced HCO3–. Internal
production of HCO3– by the hydration of CO2, also produces H+
ions that are released from the tissues, transported by the blood to
excretion sites on the epithelial membrane, and can be exchanged
for Ca2+. During times of Ca2+ depletion, Ca2+ can be acquired from
the shell, taken into the soft tissues and blood for transport to sites
in need to Ca2+ supplement. Thus in adult Lymnaea, Ca2+ is
transported between ambient media, fresh tissues, and shell via the
blood (Greenaway, 1971). Considering that these embryos complete
direct development, hatching as fully developed snails, it is not
surprising to find that the method for acquisition of shell forming
ions in embryos apparently is similar to that modeled by Greenaway
(Greenaway, 1971).
When applying pharmacological agents that were developed and
tested for use on mammals, it is necessary to consider that there
may be a reduced degree of specificity of the drug. Additionally,
using these chemicals with ion-sensitive microelectrodes can
interfere with the sensitivity and function of the electrode. However,
our preliminary tests for drug interaction and/or poisoning of the
electrode, and the general agreement between results obtained by
the two techniques used in the present study, give confidence in the
accuracy of our findings. The voltage-dependent Ca2+ channel
blockers and bafilomycin gave consistent results between whole egg
mass net flux measurements and assessment of flux rates by
microelectrodes validating the reported observation. With the
exception of EIPA, none of the drugs interfered with microelectrode
function at the concentrations used in our experiments.
Unfortunately, EIPA interfered with both Ca2+- and H+-selective
microelectrodes. EIPA concentrations of 100moll–1 reduced the
slope of the Ca2+-selective microelectrodes and caused the H+
microelectrode voltage to drift. These effects are consistent with a
poisoning of the ionophore cocktail by EIPA at 100moll–1. We
could not use SIET, therefore, to verify the whole egg mass flux
observations that suggested the presence of a cation/H+ exchanger.
Nevertheless, the excellent agreement between observations when
made with both techniques is reassuring. L-type voltage-dependent
Ca2+ channels and an EIPA-sensitive cation exchanger appear to be
involved in Ca2+ uptake in post-metamorphic embryos (Fig.9).
In the absence of external HCO3–, Lymnaea stagnalis embryos
grow, form shell, develop normally, and hatch in the same time
intervals as controls (Ebanks et al., 2010). These observations point
to an internal source of HCO3–/CO32– for shell formation via the
hydration of endogenous CO2. For this hydration reaction to allow
for CO32– accumulation within the organism and for shell formation,
hydrogen ions resulting from the hydration reaction must be
eliminated (Fig.9). We have demonstrated that the enzyme carbonic
anhydrase (CA), which facilitates the hydration of endogenous CO2
is involved in the acquisition of ions for calcification. Also, though
THE JOURNAL OF EXPERIMENTAL BIOLOGY
Embryonic snail shell ion acquisition
Ca2+ net flux (µmol g–1 h–1)
0.8
Hemolymph
0.6
0.4
~
Ca2+
VDCC
Ca2+
0.2
(7)
0
–0.2
(7)
–0.4
*
0.8
TA net flux (µmol g–1 h–1)
Water
A
4097
B
(7)
Ca2+
?
H+ + HCO3–
H+
0.6
0.4
2H
+
HCO3–
CO2 +H2O
CO2
CA
~
?
?
HCO3–
–
0.2
0
(7)
–0.2
*
–0.4
Control
EIPA
Fig.8. Comparison of net Ca2+ (A) and titratable alkalinity (B) flux rates
(molg–1h–1) under control conditions (0.1% DMSO) and during treatment
with 100moll–1 EIPA. Values are means ± s.e.m. Numbers in
parentheses are the numbers of egg masses examined. Ambient
[Ca2+]838±16moll–1. *, significantly different from control (P<0.05).
developing snails show an apparent net uptake of Ca2+ and HCO3–
equivalents, the movement of acid–base equivalents is in reality a
combination of acid secretion and base uptake, because it persists,
although at reduced rates, in absence of ambient HCO3– (Ebanks et
al., 2010). Our SIET measurements of H+ gradients further confirm
the role of H+ excretion.
The effects of CA inhibition indicated a direct role for CA in
carbonate production in post-metamorphic embryos but also points
to an indirect role in Ca2+ acquisition that is revealed when
considered in conjunction with the bafilomycin A1 results.
Bafilomycin inhibits V-ATPase activity, which has been found to
be ubiquitous among eukaryotes (Harvey, 1992; Finbow and
Harrison, 1997; Harvey et al., 1998). This includes invertebrates
and more directly related to our model species, calcifying organisms
such as blue crabs (Cameron and Wood, 1985). This enzyme has
also been characterized in tissues of freshwater bivalves Anodonta
cygnea (Machado et al., 1990; da Costa et al., 1999; Oliveira et al.,
2004), Unio complanatus (Hudson, 1993) and Nucella lamellosa
(Clelland and Saleuddin, 2000). The connection between H+-pump
activity and cation uptake has been demonstrated in epithelia of a
variety of species (Harvey et al., 1998), including Ca2+ uptake by
post-moult blue crabs Callinectes sapidus (Cameron and Wood,
1985; Neufeld and Cameron, 1992). There have also been reviews
on the presence of this pathway in crustaceans (Neufeld and
Cameron, 1993). Furthermore, H+-pump-driven active cation uptake
has been documented in adults of this species (Ebanks and Grosell,
2008) as well as in several other freshwater species including carp
(Fenwick et al., 1999), tilapia (Fenwick et al., 1999) and trout (Bury
and Wood, 1999; Grosell and Wood, 2002; Lin and Randall, 1991).
Thus inhibition of Ca2+ uptake in the presence of the V-ATPase
Fig.9. A schematic diagram of proposed mechanisms for Ca2+ and HCO3–
acquisition in an epithelial cell of a post-metamorphic embryo of the
freshwater common pond snail Lymnaea stagnalis. H+ and HCO3– are
produced by carbonic anhydrase-catalyzed hydration of CO2. The H+ ions
are excreted via the H+-pump (left-hand closed circle with ~), which
contributes to maintenance of an inside negative electrical gradient, driving
Ca2+ uptake through voltage-dependent Ca2+ channels (VDCC). In addition,
H+ excretion facilitates exchange of Ca2+ for H+ on an electrogenic
Ca2+/1H+ exchanger that appears to be unique to invertebrates (solid circle
with ?) (Greenaway 1971; Okech et al., 2008; Ahearn et al., 1994; Ahearn
et al., 2001). There is also evidence that uptake of HCO3–, when available
in the water, occurs via an undefined pathway (Ebanks et al., 2010). It is
assumed that a basolateral Ca2+-ATPase is responsible for transport of
Ca2+ across the basolateral membrane (right-hand closed circle with ~) and
that HCO3– ions are passed across the basolateral membrane to the
extracellular fluid or hemolymph. See text for further details.
inhibitor, bafilomycin, in developing snail embryos is persistent
across later time points (up to 80% reduction in post-metamorphic
Ca2+ uptake) and appears to be consistent with investigations in other
species. Thus the H+ pump plays a significant role in Ca2+ flux in
post-metamorphic stages. By electrogenic excretion of H+ ions, the
electrical potential across the apical membrane, and thereby the
electrochemical gradient for Ca2+ uptake is maintained. In addition,
the observations of reduced acid excretion and Ca2+ uptake in the
presence of the specific Na+/H+ exchange inhibitor, EIPA, points
to an even more direct coupling of H+ extrusion to Ca2+ uptake.
However, Hudson found no effect of 1mmoll–1 amiloride or Na+depleted solutions on acid secretion in Ussing-type chamber
experiments on mantle epithelia of U. complanatus (Hudson, 1993).
EIPA was not included in the study, but there was no detectable
coupling between a proxy for acid secretion, cAMP-dependent shortcircuit current (ISC) (Machado et al., 1990), and unidirectional Na+,
K+, Ca2+ or Cl– fluxes in the same species (Hudson, 1993).
Nevertheless, our data suggest the presence and involvement of a
Ca2+/H+ exchanger in L. stagnalis. It is interesting to note that
electrogenic Ca2+/H+ exchange would be facilitated by parallel
electrogenic H+ extrusion by the H+ pump.
Taken together, our results suggest that through the use of the
ionic products of CO2 hydration, H+ and HCO3–, the high Ca2+ and
carbonate demands for shell formation in freshwater are met and
maintained with notable efficiency. The higher the rate of CO2
THE JOURNAL OF EXPERIMENTAL BIOLOGY
4098 S. C. Ebanks, M. J. O’Donnell and M. Grosell
hydration, the higher the rate of HCO3– formation, and the higher
the amount of H+ excretion by the proton pumps and/or cation
exchange, which in turn provides for Ca2+ uptake. Inhibition of CA
activity leads to inhibition of transport of both shell-forming ions.
Admittedly, inhibition of the ability to hydrate respiratory CO2 can
have broader impacts on the physiological level. However, when
the results of CA inhibition are taken in conjunction with the
response to H+ pump and NHE inhibition as well as the ability of
the embryos to develop in HCO3–-free conditions (Ebanks et al.,
2010), there is substantial evidence that endogenously produced
carbonate can be an exclusive carbonate source for these postmetamorphic embryos. A further point to consider is that in addition
to protons produced from hydration of metabolic CO2, H+ ions are
liberated by the calcification process (HCO3–+Ca2+rCaCO3+H+)
adding to the need for acid secretion by these animals. By coupling
Ca2+ uptake and HCO3– production with the generation of inside
alkaline conditions via H+ extrusion, the conditions for calcification
within the egg and egg mass are enhanced. Furthermore, the
depletion of Ca2+ within the perivitelline fluid and gelatinous matrix
(tunica interna) between the eggs to levels below water
concentrations during later embryonic stages (Ebanks et al., 2010)
creates an inward Ca2+ gradient and thus can enhance the influx of
Ca2+ from the surrounding water while decreasing the energy
required for ion uptake.
Conclusions
Acquisition of the necessary Ca2+ and carbonate for calcification
by embryos of the freshwater common pond snail Lymnaea stagnalis
are intricately intertwined. These embryos take up Ca2+ from
ambient media after maternal stores in the perivitelline fluid have
been depleted; but they also directly utilize endogenously produced
bicarbonate from the CA-catalyzed hydration of CO2. Protons
generated from CO2 hydration are excreted, which fuels continued
Ca2+ uptake and allows for accumulation of bicarbonate and
maintenance of alkaline internal conditions that are favorable for
calcification. With endogenous production of HCO3– for
calcification perpetuating continued Ca2+ uptake, these embryos are
capable of efficient calcification under low ionic strength conditions.
With global climate change, studies are necessary to determine
how ambient pH and bicarbonate buffering affect the ability of these
freshwater snails to calcify. Furthermore, evaluating the extent to
which acid secretion rather than base uptake is the basis for
calcification in other freshwater as well as marine calcifiers offers
an exciting area for comparative studies.
LIST OF ABBREVIATIONS
CA
EIPA
ETOX
NHE
carbonic anhydrase
ethylisopropylamiloride (C11H18ClN7O)
ethoxzolamide (C9H10N2O3S2)
Na+/H+ exchanger
ACKNOWLEDGEMENTS
S.C.E. appreciates the technical assistance and hospitality of Andrea Kocmarek
and Erin Leonard of McMaster University offered during her visit to the O’Donnell
laboratory. This research was funded in part by the NOAA Educational
Partnership Program, Environmental Cooperative Science Center and the
RSMAS Marine Science Graduate Student Organization Student Travel Fund.
REFERENCES
Ahearn, G. A., Zhuang, Z., Duerr, J. and Pennington, V. (1994). Role of invertebrate
electrogenic 2Na+/1H+ antiporter in monovalent and divalent cation transport. J. Exp.
Biol. 196, 319-335.
Ahearn, G. A., Mandal, P. K. and Mandal, A. (2001). Biology of the 2Na1+/1H1+
antiporter in invertebrates. J. Exp. Zool. 289, 232-244.
Baldwin, E. (1935). The energy sources in ontogenesis. VIII. The respiratory quotient
of developing gastropod eggs. J. Exp. Biol. 12, 27-35.
Bielefeld, U. and Becker, W. (1991). Embryonic development of the shell in
Biompharia glabrata (Say). Int. J. Dev. Biol. 35, 121-131.
Bury, N. R. and Wood, C. M. (1999). Mechanism of branchial apical silver uptake by
rainbow trout is via the proton-coupled Na+ channel. Am. J. Physiol. Regul. Integr.
Comp. Physiol. 277, R1385-R1391.
Cameron, J. N. and Wood, C. M. (1985). Apparent H+ excretion and CO2 dynamics
accompanying carapace mineralization in the blue crab (Callinectes sapadus)
following moulting. J. Exp. Biol. 114, 181-196.
Clelland, E. S. and Saleuddin, A. S. M. (2000). Vacuolar-type ATPase in the
accessory boring organ of Nucella lamellosa (Gmelin) (Mollusca: Gastropoda): Role
in shell penetration. Biol. Bull. 198, 272-283.
da Costa, A. R., Oliveira, P. F., Barrias, C. and Ferreira, H. G. (1999). Identification
of a V-type proton pump in the outer mantle epithelium of Anodonta cygnea. Comp.
Biochem. Physiol. 123A, 337-342.
Donini, A. and O’Donnell, M. J. (2005). Analysis of Na+, Cl–, K+, H+ and NH4+
concentration gradients adjacent to the surface of anal papillae of the mosquito
Aedes aegypti: application of self-referencing ion-selective microelectrodes. J. Exp.
Biol. 208, 603-610.
Ebanks, S. C. and Grosell, M. (2008). Fluid and osmolyte recovery in the common
pond snail Lymnaea stagnalis following full-body withdrawal. J. Exp. Biol. 211, 327336.
Ebanks, S. C., O’Donnell, M. J. and Grosell, M. (2010). Acquisition of Ca2+ and
carbonate for shell formation in embryos of the common pond snail Lymnaea
stagnalis. J. Comp. Physiol. B 180, 953-965.
Fenwick, J. C., Wendelaar Bonga, S. E. and Flik, G. (1999). In vivo bafilomycinsensitive Na(+) uptake in young freshwater fish. J. Exp. Biol. 202, 3659-3666.
Finbow, M. E. and Harrison, M. A. (1997). The vacuolar H+-ATPase: a universal
proton pump of eukaryotes. Biochem. J. 324, 697-712.
Flik, G., Verbost, P. M. and Wendelaar Bonga, S. E. (1995). Calcium transport
processes in fishes. In Cellular and molecular approaches to fish ionic regulation
(ed. C. M. Wood and T. J. Shuttleworth), pp. 317-336. New York: Academic Press.
Greenaway, P. (1971). Calcium regulation in the freshwater mollusc Limnaea stagnalis
(L.) (Gastropoda: Pulmonata). II. Calcium movements between internal calcium
compartmants. J. Exp. Biol. 54, 609-620.
Grosell, M. and Wood, C. M. (2002). Copper uptake across rainbow trout gills:
mechanisms of apical entry. J. Exp. Biol. 205, 1179-1188.
Grosell, M., Blanchard, J., Brix, K. V. and Gerdes, R. (2007). Physiology is pivotal
for interactions between salinity and acute copper toxicity to fish and invertebrates.
Aquat. Toxicol. 84, 162-172.
Hagiwara, S. and Byerly, L. (1981). Calcium channel. Annu. Rev. Neurosci. 4, 69125.
Harvey, W. R. (1992). Physiology of V-ATPases. J. Exp. Biol. 172, 1-17.
Harvey, W. R., Maddrell, S. H. P., Telfer, W. H. and Wieczorek, H. (1998). H+ VATPases energize plasma membranes for secretion and absorption of ions and
fluids. Amer. Zool. 38, 426-441.
Hudson, R. L. (1993). Bafilomycin-sensitive acid secretion by mantle epithelium of the
freshwater clam, Unio complanatus. Am. J. Physiol. Regul. Integr. Comp. Physiol.
264, R946-R951.
Lin, H. and Randall, D. (1991). Evidence for the presence of an electrogenic proton
pump on the trout gill epithelium. J. Exp. Biol. 161, 119-134.
Machado, J., Ferreira, K. G., Ferreira, H. G. and Fernandes, P. L. (1990). The acidbase balance of the outer mantle epithelium of Anodonta cygnea. J. Exp. Biol. 150,
159-169.
Marshall, W., Bryson, S. and Wood, C. (1992). Calcium transport by isolated skin of
rainbow trout. J. Exp. Biol. 166, 297-316.
Marxen, J. C., Becker, W., Finke, D., Hasse, B. and Epple, M. (2003). Early
mineralization in Biomphalaria glabrata: Microscopic and structural results. J. Mollus.
Stud. 69, 113-121.
Morrill, J. B. (1982). Development of the pulmonate gastropod, Lymnaea. In
Developmental Biology of Freshwater Invertebrates (eds. W. Harrison and R. F.
Cowdon), pp. 399-483. New York: Alan R. Liss.
Neufeld, D. S. and Cameron, J. N. (1992). Postmoult uptake of calcium by the blue
crab (Callinectes sapadus) in water of low salinity. J. Exp. Biol. 171, 283-299.
Neufeld, D. S. and Cameron, J. N. (1993). Transepithelial movement of calcium in
crustaceans. J. Exp. Biol. 184, 1-16.
Nitschke, R., Fröbe, U. and Greger, R. (1991). Antidiuretic hormone acts via V1
receptors on intracellular calcium in the isolated perfused rabbit cortical thick
ascending limb. Pflügers Arch. 417, 622-632.
Okech, B. A., Boudko, D. Y., Linser, P. J. and Harvey, W. R. (2008). Cationic
pathway of pH regulation in larvae of Anopheles gambiae. J. Exp. Biol. 211, 957968.
Oliveira, P. F., Lopes, I. A., Barrias, C. and Rebelo da Costa, A. M. (2004). H+ATPase of crude homogenate of the outer mantle epithelium of Anodonta cygnea.
Comp. Biochem. Physiol. 139A, 425-432.
Perry, S. F. and Flik, G. (1988). Characterization of branchial transepithelial calcium
fluxes in freshwater trout, Salmo gairdneri. Am J. Physiol. Regul. Integr. Comp.
Physiol. 254, R491-R498.
Perry, S. F., Shahsavarani, A., Georgalis, T., Bayaa, M., Furimsky, M. and
Thomas, S. L. Y. (2003). Channels, pumps, and exchangers in the gill and kidney of
freshwater fishes: Their role in ionic and acid-base regulation. J. Exp. Zool. 300A,
53-62.
Poncet, V., Merot, J. and Poujeol, P. (1992). A calcium-permeable channel in the
apical membrane of primary cultures of the rabbit distal bright convoluted tubule.
Pflügers Arch. 422, 112-119.
Shahsavarani, A. and Perry, S. F. (2006). Hormonal and environmental regulation of
epithelial calcium channel in gill of rainbow trout (Oncorhynchus mykiss). Am J.
Physiol. Regul. Integr. Comp. Physiol. 291, R1490-R1498.
THE JOURNAL OF EXPERIMENTAL BIOLOGY